DK157061B - PROCEDURE FOR PREPARING A HEPARINOID WITH ANTITHROMOBOTIC EFFECT - Google Patents

Abstract

A new anti-thromboticum based on a mixture of oligo- and polysaccharides is provided. The invention also relates to methods for preparation thereof and to pharmaceutical compositions including the said anti-thromboticum. The new product is of natural origin and can be obtained from mammal tissue, particularly intestinal mucous. The product is useful because of its high "benefit/risk" ratio (i.e. the ratio between the anti-thrombotic activity and the haemorrhagic activity).

Description

DK 157061 B

The present invention relates to a process for the preparation of a novel heparinoid having antithrombotic activity on the basis of a mixture of oligo- and polysaccharides, having the composition set forth in claim 1 and having the properties stated at 5 sites.

The method according to the invention is peculiar to the characterizing part of claim 1.

It is known that certain mucopolysaccharides affect blood coagulation properties. The best mucopolysaccharide is heparin, a sulfated mucpolysaccharide used to prevent and treat venous thrombosis and thromboembolism. The anti-thrombotic action of heparin is based on acceleration of the inhibition of blood coagulation factors by the aid of anti-thrombin III. A major difficulty in using heparin to prevent and treat thrombosis and thromboembolism is the bleeding-inducing ability of heparin. By optimizing the dosage method and frequency used, the risk of bleeding can be reduced to some extent, but the real association between the anti-thrombotic and heparin properties of the heparin cannot be affected.

As a result, the prophylactic use of heparin 25 has been limited to those indications in which the coagulation system is activated only moderately, as in the case of mild forms of deep vein thrombosis. A further deficiency of heparin is its relatively short duration of action, so that to achieve effective prevention, a dose of 30 doses should be administered at least twice daily.

Numerous attempts have already been made to prepare "enhanced heparins" or to produce heparinoids with improved properties. British Patent No. 2,002,406 relates to e.g. an oligo-heteropolysaccharide mixture having a molecular weight distribution between 2000 and 5000 daltons, which is obtained by depolymerization of heparin and / or isolated from the mother liquor from the normal heparin preparation, after which the substance obtained is sulfated.

DK 157061 B

It is argued that the ratio of the anti-thrombotic (in vivo) effect to the anti-coagulation (in vitro) effect of the substance thus obtained is more favorable than for the hepari n.

5

A novel anti-thrombotic active heparinoid, which is a mixture of oligo and heteropolysaccharides, has now been found, with a striking dissociation between the anti-thrombotic and hemorrhagic effect (haemorrhagic ability).

10

Under various bleeding studies in rats (muscle-bleeding test, capillary bleeding test), the novel heparinoid does not appear to cause any bleeding or hardly cause more bleeding than a placebo, while the hemorrhagic effect over a wide dose range (10/250 mg kg) grew only slightly. The anti-thrombotic effect per Although unit weight is inferior to heparin, the "benefit / risk" ratio (i.e., the ratio of anti-thrombotic and hemorrhagic) is 10-40 times more favorable than heparin. This will be explained in more detail below by means of comparative examples.

The novel heparinoid having antithrombotic effect is of natural origin and consists of a mixture of oligo and polysaccharides based on hexose derivatives such as glucuronic acid, iduronic acid, glucosamine, galactosamine and sulfated and acetylated derivatives thereof. The novel product is a white, amorphous, slightly hygroscopic powder with rim properties: a) a molecular weight distribution (determined by comparison with dextran by gel permeation chromatography on a macropores silica matrix (Nucleosil 50-5, Nucleosil Denmark unregistered trademark (with 0.5 molar ammonium acetate buffer (pH 35 = 5.0) as eluant) between 2,000 and 40,000 daltons with a main peak between 2,500 and 15,000 daltons, more specifically between 4,000 and 10,000 daltons and an average of between 5,000 and 8,000 daltons and usually an extra top and / or shoulder in 3

DK 157061 B

the range between 15,000 and 60,000 daltons, more particularly between 30,000 and 50,000 daltons, with an average molecular weight of about 40,000 daltons; + 30 ° and + 70 °, c) a nitrogen content between 1.5 and 4% by weight, preferably 10 between 2.5 and 3.5% by weight, d) a sulfur content between 5 and 7.5% by weight, preferably between 5.5 and E) content of ionic group in meq / g between 3 and 5, preferably between 3.5 and 4.5; f) content of suifamide groups in meq / g between 0.5 and 1.5; preferably between 0.5 and 1.0, 20 g) a glucosamine content in meq / g of 0.5 - 1.5, h) a galactosamine content in meq / g of 0.0 - 0.6, i) an idose (iduronic acid) / g 1ucose (glucuronic acid) ratio of 0.5 - 3, more particularly 1-3.

Although it is not possible to accurately specify the composition of the novel oligo and polysaccharide, the above-mentioned parameters characterize the product to a sufficient degree, especially in relation to its pharmacological profile.

The pharmacological profile of the novel heparinoid is characterized by: 1) an anti-coagulation effect (US P) of less than 10 international units per mg (IU / mg), thus only a fraction (usually less than 5%) of the size of heparin USP, 35 4

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2) a negligible anti-thrombin effect (less than 1% of the effect of hepari n USP); 3) an anti-X effect of less than 20% of the effect of heparin, usually between 2.5 and 15%; 4) an anti-thrombotic activity (Umetsum model) with an ID 50 of about 2-8 mg / kg iv (ID50 of heparin USP is about 0.4-0.5 mg / kg iv), 5) a hemorrhage effect that rises sharply over a wide dose range (up to 300 mg / kg iv) and a dose of 1 mg / kg iv tufts can be seen and grow rapidly at higher doses; 6) a "benefit-risk" ratio which is 10-40 times more favorable than the ratio of heparin USP to the anti-thrombotic effect compared to the hamorrhagic effect, and 7) a half-life which is certainly twice as long as the half-life of heparin USP.

This interesting profile makes the novel product unsuitable for the prevention and treatment of vein thrombosis and thromboembolism. This product proves particularly suitable for the prevention of DVT (deep vein thrombosis) in patients who have undergone or are undergoing hip surgery. So far, no adequate preventive agents have been used against the frequent occurrence of DVT and kidney embolisms associated with hip surgery. Low doses (s.c.) of heparin are not effective, and higher i.v. doses are not advisable due to the high risk of bleeding.

35 The novel product is found to be without mutagenic and toxic properties even in high doses (up to 400 mg / kg / day).

The new product can be obtained in various ways. Mammalian tissues, such as lungs, pancreas, liver, intestines and saliva 6

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cellular intestinal mucus can serve as a basic material. The product is first released from the mammalian tissue by autolysis or by proteolytic enzymes (eg, pancreatic or bacterial enzymes, such as Bacillus subtilis proteases).

The product is then isolated by precipitation with methanol.

Other methods of isolation are to bind the product to a quaternary, aliphatic ammonium base or a basic ion exchanger and then elute the product with aqueous saline solutions. Further purification of the product may be effected by fractional precipitation with methanol.

Further studies have shown that by treating the product thus obtained with chondroitinase ABC, the 15 major portion of the high molecular weight galactosamine-containing polysaccharides present are removed. However, the remaining product (70-75% of the product initially) appears to have almost the same surprising pharmacological profile as the untreated product; in the said enzymatic treatment, 20 in other words, a predominantly high molecular weight inactive fraction (25-30% by weight) is removed.

The physical properties of this product obtained after treatment with chondroitinase ABC remain within the characteristics set forth above for the novel heparinoid, except that in the molecular weight distribution, the peak and / or shoulder of the high molecular weight fraction has almost disappeared. the specific rotation is found to increase to a value between + 45 ° + 75 °, and the galactosamine content is practically reduced to 0. It is clear that the invention also encompasses this product.

The novel product or products may be prepared in the manner commonly used for heparin into a pharmaceutical dosage form, e.g. by solution in water suitable for injection, to which, if necessary, additional pharmaceutically acceptable excipients (preservative, certain salts) have been added. Clinical application happens at

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6 by subcutaneous or intravenous (possibly alternating) injection or by infusion. Other dosing methods can also be used, such as intra-pulmonary application via spray inhaling or administration using a suppository.

5

The invention will be further described by the following examples.

Example I

10

Bovine lung (100 kg) was treated with porcine pancreas proteolytic enzymes. After 15 hours of incubation at pH 8.5 and a temperature of 40 ° C, the mixture was filtered off. The clear filtrate was contacted with a strong basic ion exchanger 15 (QAE Sephadex® A50) and stirred for 15 hours. :

The ion exchanger was filtered off and eluted with an aqueous solution of NaCl (200 g / l). Methanol was added to the eluate until 50% v / v. The resulting precipitate was removed, then methanol was added to the mother liquor until 75% v / v. The precipitate was recovered, washed with 100% methanol and terraced. The obtained amorphous white powder (22.7 g) had a galactosamine content of 0.45 mmol / g, a glucosamine content of 0.54 mmol / g, and an average molecular weight of 6,600 daltons with an additional peak at about 38,000 daltons ( determined with respect to dextran), a

[a] 20-value of + 34.2®, a sulfur content of 5.7%, a nitrogen-D

content of 2.6%, content of ionic groups of 3.90 meq / g, content of sulphamide groups of 0.69 mmol / g and an idose / glucose ratio of 2.1.

The electrophoresis pattern of the product is shown in FIG. 1 (agarose gel, equilibrated in 0.2 molar calcium acetate, pH = 7.2, buffer 0.2 molar calcium acetate, 300 V max, 7 mA, 30 minutes.

35 7

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Example II

Pig mucosa (100 kg) was treated with proteases of Baci1-lice subtilis at 35 ° C and a pH of 8.2 for 24 hours. The mixture was filtered and the clear filtrate worked up similarly as described in Example 1. The yield was 3.2 g of white amorphous powder with a galactose amine content of 0.38 mmol / g, a glucosamine content of 0.82. mmol / g, an average molecular weight of 6,100 daltons with an additional peak at an average of 42,000 daltons (determined with respect to dextran), a [a] 20

D

35.1 °, sulfur content of 5.9%, nitrogen content of 2.7%, content of ionic groups of 3.70 meq / g, content of 15 sulfamide groups of 0.73 mmol / g and an idose / glucose ratio of 1.9.

The product had a nuclear magnetic resonance spectrum (4% in O₂O at 50 ° C, 270 MHz) as shown in FIG. 2nd

20

Example III

Bovine intestinal mucus (10 m 3) was worked up with pancreatin (40%, pH)

8.5, 20 hours). The mixture was filtered and the filtrate worked in the manner described in Example I. The yield was 335 g of a white amorphous powder with a galactosamine content of 0.28 mmol / g, a glucosamine content of 0.90 mmol / g, an average molecular weight of 5,600 daltons with a further peak at an average of 41,000 daltons ( determined with respect to dextran), a 30 I *) 20

D

a value of + 36.9 °, a sulfur content of 6.1%, a nitrogen content of 2.8%, a content of ionic groups of 4.0 meq / g, a content of sulfamide groups of 0,75 mmol / g and a idose / glucose ratio of 1.8.

35 “8

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Example IV

The product of Example III (100 g) was treated with chondroitininase ABC. Via methanol precipitation (50-75% v / v), the remaining product was isolated. 72 g of product (white, amorphous powder) with a galactosamine content of 1.20 mmol / g was obtained, an average molecular weight of 5,400 daltons with an extremely small additional peak at an average of 40,000 daltons (determined with respect to dextran), a [ a] 20 value of 10 D

+ 61.2 °, a sulfur content of 6.2%, a nitrogen content of 2.75%, a content of ionic groups of 4.1 meq / g, a content of sulphamide groups of 0.91 mmol / g and a idose / glucose ratio of 1.9.

15

Pharmacological studies

The products obtained in Examples 1-4 were subjected to a series of pharmacological studies in comparison with Hepherin USP.

Impact on blood coagulation

Product Anti-coagulation Anti-drum Anti-Xa 25 action effect effect (IU / mg) (IU / mq) _ (IU / mq)

Heparin 179 164 167

Example I 1 0.5 4.5 "II 1 0.1 5" III 3 1.0 6 "IV 4 1.3 8 30 _______

The anti-coagulation effect was determined according to the USP method. The anti-thrombin effect and anti-XA effect were determined using a chromogenic substrate method using purified quantum thrombin bin III and the substrates (Kabi AB, Sweden) S-2238 and S-2222, respectively.

The results in the table show that the products of Examples I-IV exhibit inhibitory activity against activated compounds.

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gulation factors with a high specificity, ie they have an anti-Xa virus greater than the anti-thrombi effect. Contrary to the product of the invention, heparin does not exhibit any specific inhibition directed against one of these coagulation factors. Thus, heparin exhibits a strong inhibition of overall coagulation, while the product of the present invention is modest and thus presents a minimal risk of bleeding.

10

Anti-thrombotic effect

Anti-thrombotic activity was determined by the Umetsumo subset (Thrombos. Haemostas. 3J [, 74-83, 1978) in rats. In this model, thrombi are provoked in arterio-venous shunts by allowing blood to flow along a silk thread for 15 minutes. The placebo effect and the effect of the substances to be investigated are measured at different doses on the thrombus formation, for the relationship between the dose and the inhibition of the thrombus effect with respect to the placebo effect it is possible to derive an ID 50 value (dose necessary for 50 % inhibition of drum formation).

25 results

Product id50 1 m9 / kg heparin USP 0.5

Example I 6.0 30 "II 5.0" III 4.0 "IV 5.3 35

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1 °

Hamorrhagic activity (a) Capillary blood test in rats 5 Placebo or the products to be examined were dosed via the dorsal ointment of the penis of anesthetized rats. After a minute, a strip of skin was manually stripped from the shaved abdomen along two pre-cut sections. The wound was covered with a gauze bandage and the skin strip was put back over the gauze.

After 10 minutes, the gauze was removed and the blood contained therein was extracted with 20 ml of water. The hemoglobin concentration in the water was measured spectrophotometrically and used as a parameter for blood loss (see Thrombos. Haemostas. 42, 466, 1979).

15

During this study, the product of Example II and heparin were compared at different doses, taking into account the difference in anti-thrombotic activity between the new product and heparin. The results are shown in FIG. 3. Similar results were obtained from the products of Examples I, II and IV.

b) Muscle blotting tests in rats 25 Biceps femoris in each paw of an anesthetized rat were excised longitudinally with a scalpel for 1 minute after the placebo or product to be examined had been administered intravenously via the dorsal ointment of the penis. Each wound was covered with a gauze bandage. After 30 minutes, the bandages became feather-30 nets. Blood loss was determined in the same way as in the capillary bleeding test. The product of Example n and heparin were compared in this test. The results are shown in FIG. 4. Similar results were obtained with the products of Examples I, III and IV.

Figures 3 and 4 clearly show the much higher tendency for heparin to bleed, whereas the tendency does not differ or hardly deviate significantly from placebo over a wide dose interval.

Claims (2)

A process for preparing a heparinoid having anti-thrombotic activity based on residues of glucuronic acid, iduronic acid, glucosamine and galactosamine and sulfated and acetylated derivatives thereof, which has the following properties: a) a molecular weight between 3.32 x 10 "21 0g 6.64 x 10-20g (2000- 40,000 daltons), with a head peak at a molecular weight of between 4,415 x 10“ 21 and 2.49 x 10-20g (2500- (15,000 daltons) and optionally an additional top and / or shoulder where the molecular weight is between 2.49 x 10 1020 and 9.96x1020g (15,000-30 60,000 daltons) (b) a specific rotation ([. -Un * 25 "and * 80 ·," (c) nitrogen content of between 1.5 and 4% by weight, 35 d) a sulfur content of between 5 and 7.5% by weight, e) a content of ionic groups of between 3 and 5 meq / g, DK 157061 B \ f) a content of sulfamide groups between 0.5 and 1.5 meq / g, g) a glucosamide content of 0.5-1.5 meq / g, 5 h) a galactosamine content of 0.0 -0.6 meq / g, i) an idose (iduronic acid) / glucose (gluc 0.5-3, 10 j) an anticoagulation activity (USP) of less than 10 international units per (k) a minimum thrombin activity (less than 1% of the heparin USP value), 15 l) an anti-X activity of less than 20%, usually 2.5-15% of the heparin equivalent value, m) a IDsg value for antithrombotic activity (Umetsu model) 20 at approx. 2-8 mg / kg iv, n) the bleeding activity hardly increases within a large dosage range (up to 300 mg / kg iv), 25 o) the benefit-risk ratio is 10-40 times more favorable than that of heparin-USP for (p) the half-life is certainly twice as long as that of 30 heparin USP, characterized in that mammalian tissues such as the lungs, pancreas, liver, intestine and intestinal mucosa are treated by autolysis or with proteolytic enzymes, filtration of the resulting mixture, isolation of the heparinoid by fractional precipitation by adding to the filtrate a maximum of 50 volumes of methanol, removal of the precipitate, further addition of no more than 75 volumes of methanol and isolation of the thus obtained. GB 157061 B the precipitation, or treatment of the filtrate with a quaternary ammonium base or a basic ion exchanger, elution of the separated base or ion exchanger with an aqueous saline solution, and treatment of eluate one by the above fractionated precipitation. A method according to claim 1, characterized in that the isolated product is subjected to the action of chondroitinase ABC to remove most of the high molecular weight polysaccharides and the remaining product is ice-blended. 15 20 25 30% 35